Mutations in the human EFNB1 gene lead to a disease known as craniofrontonasal syndrome (CFNS), which involves a number of craniofacial anomalies including craniosynostosis, frontonasal dysplasia, severe hypertelorism, and cleft palate. Mutating efnB1 in mice causes similar defects, making these mice a good model for the human disease. EFNB1 is X-linked in both mice and humans; consequently, hemizygous mutant males have no functional ephrin-B1, while heterozygous mutant females are mosaic for cells capable of expressing ephrin-B1 due to random X-inactivation. However, heterozygous females have much more severe phenotypes than either hemizygous null males or homozygous null females. This phenomenon is thought to be related to ephrin-B1-mediated segregation of cells into large ephrin-B1-positive and -negative patches. In this proposal, I aim to explore the mechanisms underlying this segregation. Ephrin-B1 is a cell-bound signaling molecule that acts in part by activating EphB receptor tyrosine kinases in a process known as forward signaling. EphB receptors can stimulate signaling through Rho family GTPases and the Ras/ERK pathway and also prompt cleavage of E-cadherin by ADAM family metalloproteases. All three of these mechanisms have been proposed to mediate cellular segregation in various developmental contexts; however, it is unclear which mechanism is at work in CFNS. In addition to forward signaling, ephrin- B1, as a transmembrane molecule, can also reverse signal back into its own cells; reverse signaling has been observed to be required for cell segregation in some contexts. My preliminary data suggest that this is not the case in CFNS. My data also indicate that activation of RhoA and the Rho-dependent kinase ROCK are critical for segregation and that segregation begins early in development. In the first aim, I propose to determine whether reverse signaling is necessary and sufficient to cause segregation using a reverse-signaling dead allele of efnB1. I will also investigate the contribution of forward signaling by ephrin-B1's receptors EphB2 and EphB3 to segregation by testing whether genetic loss of forward signaling capacity can rescue the sorting phenotype in heterozygous efnB1+/- mice. In the second aim, I will identify the downstream signaling pathways involved in cell sorting using a pharmacogenetic approach in HEK293 cell culture. Based on my preliminary data, I will focus on components of Rho family GTPase signaling pathways. I will then examine segregating cells in culture and embryos for activation of Rho and other potential components of the EphB-Rho signaling pathway. Finally, I will validate these findings in a roller bottle embryo culture assay system tha I have established, in which embryos are isolated before segregation begins and cultured in roller bottles until a point at which segregation is normally readily apparent. The results of thi study will illuminate a poorly understood congenital condition and contribute to a better understanding of fundamental Eph/ephrin signaling mechanisms.